Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is etiologically linked to several human cancers, including AIDS-associated B-cell lymphomas. Previously, we have reported that both EBV-encoded microRNAs (miRNAs) and EBV-induced cellular miRNAs play a critical role in promoting cellular transformation by EBV in culture. Here, we propose an integrated series of experiments designed to lead to a mechanistic understanding of how the miRNAs encoded by, or induced by, EBV infection contribute to the growth and transformation of human cells in culture and in vivo. We will dissect the mechanisms used by EBV-encoded and EBV-induced cellular miRNAs to facilitate the formation of stably immortalized lymphoblastoid cell lines (LCLs) and, more generally, examine how miRNAs contribute to lymphomagenesis and control the decision between viral latency and lytic replication. In addition, we will determine whether specific viral or cellular miRNAs promote the growth and viability of latently EBV-infected diffuse large B cell lymphoma cells (DLBCLs) and nasopharyngeal carcinoma (NPC) cells, in order to ascertain whether EBV uses common mechanisms to transform B lymphocytes and epithelial cells. While one important focus of this grant will be on a cluster of three EBV-encoded miRNAs (miR-BHRF1-1, 1-2 and 1-3) that is expressed at high levels in LCLs and in many DLBCLs, we will also address how the 22-miRNA EBV BART cluster affects the ability of EBV-transformed cells to grow in culture and to avoid immune elimination. Critically, we will integrate miRNA target identification, using a powerful cross-linking and immunoprecipitation (CLIP) technique, with phenotypic studies of miRNA:mRNA interactions to define the mechanisms of action used by these miRNAs to mediate EBV latency and pathogenesis.